This R21 exploratory proposal addresses a major gap in U.S. reproductive medicine, namely the lack of reliable, objective methodology for accurate diagnostics of human male infertility. Our goal is to validate the sperm-specific thioredoxin SPTRX3 as a novel biomarker of human sperm quality. We will examine how sperm SPTRX3 levels in male infertility patients correlate to a failure to conceive, to spontaneous abortion and to unwanted multiple births in their female partners treated by assisted reproductive therapies (ART). Current methods of human semen analysis and diagnostics of human male infertility rely heavily on subjective light microscopic evaluation. This is inaccurate because of a significant overlap between semen parameters of fertile and infertile men. Objective, automated methods for sperm analysis exist but are expensive and confined to specialized reference laboratories, as opposed to an inexpensive doctor's office fertility test. We have already established that SPTRX3 protein is carried exclusively by defective, morphologically deviant human spermatozoa. Our preliminary data demonstrate that the increased semen content of SPTRX3-carrying spermatozoa coincides with reduced pregnancy rates after ART. In contrast, ART couples with male partners exhibiting low semen SPTRX3 levels produced significantly more multiple births. This two year project has ONE SPECIFIC AIM, i.e. to demonstrate a statistical relationship between semen SPTRX3 levels and the occurrence of fertilization failure, spontaneous pregnancy loss and multiple pregnancies in couples from a general infertility clinic population. Our HYPOTHESIS is that the female partners of men with low SPTRX3 levels are more likely to conceive by ART and less likely to suffer a spontaneous abortion, but they are also more prone to multiple births. Semen SPTRX3 content will be evaluated in raw and gradient/swim-up purified semen samples from infertile couples by automated flow cytometric analysis and epifluorescence-light microscopic analysis of immunolabeled spermatozoa, and by semi-quantitative western blotting/densitometry technique. The influence of high semen SPTRX3 content will be evaluated against basic measures of treatment outcome, including fertilization and first embryo cleavage rates, incidence of spontaneous abortion following a successful assisted fertilization and embryo transfer, and the incidence of multiple pregnancies in couples treated by ART. To gain mechanistic understanding of how SPTRX3 carryover affects human sperm function, we will correlate sperm chromatin structure, likely affected by the presence of SPTRX3-containing nuclear vacuoles, with flow cytometric SPTRX3 data. We will also assess male pronuclear development after heterologous ICSI of sperm from high/low SPTRX3 samples into hamster ova, an assay commonly used to predict human sperm fertilizing ability and developmental potential. Based on feedback from our clinical collaborators, one of whom will participate in this project, we anticipate that the SPTRX3 based test will allow clinicians to make a treatment decision between intrauterine insemination (IUI) versus IVF/ICSI in the general infertility clinic-population. In the IVF/ICSI treated couples, a decision will be facilitated for how many embryos should be transferred to obtain a singleton pregnancy, as opposed to no pregnancy or an unwanted multiple birth. Upon validation by the proposed research, this novel biomarker can be quickly transferred to clinical practice in the form of probe for a reference laboratory test, or a lateral flow cassette (dipstick) for doctor's office use;an over the counter home fertility test could also be developed. PUBLIC HEALTH RELEVANCE: This project will explore and validate a novel biomarker of human male infertility, the recently discovered sperm borne protein SPTRX3. Preliminary data suggest that this biomarker can be used to more accurately diagnose male infertility and also to predict the likelihood of multiple births and spontaneous pregnancy losses after assisted fertilization. The proposed exploratory research is necessary for future diagnostic technology development leading to the introduction of this innovative diagnostic tool in US infertility clinics. Annually, more than 135,000 infertile couples are treated for infertility in US clinics, where the treatment-decision making still relies on outdated, highly subjective light microscopic method of semen evaluation.